Mitochondria are known to participate in a host of cellular processes such as apoptosis, heme metabolism, and the regulation of energy balance. Largely due to the mitochondrial impact on energy status, the bulk of most mitochondrial research involved liver, muscle, and pancreatic cells. Since endothelial cells are predominantly glycolytic, little effort was applied to the role of mitochondria in the endothelium, despite recen appreciation that endothelial cells can impact tissue metabolism and homeostasis. In the previous funding period, we found that endothelial PGC-1, a transcriptional co-activator for many mitochondrial genes, was critical for endothelial cell stress adaptation. In this application, we present data that uncoupling protein-2 (UCP2), a PGC-1 target gene, is particularly important for mitochondrial stress adaptation in the endothelium. In settings of tissue repair or increased fuel utilization, endothelial cells tightly regulate their mitochondrial proton gradient () via uncoupling protein-2 (UCP2), in order to prevent mitochondrial stress manifest as mitochondrial network fragmentation that promotes endothelial dysfunction. The long-term objective of this investigative program is to understand how mitochondria modulate endothelial function and how we can use this information for new therapies. In order to achieve this objective, we submit as a central hypothesis that endogenous UCP2 functions to prevent mitochondrial stress and, as a consequence, UCP2 is a major determinant of endothelial cell function and vascular homeostasis. In order to achieve this project objective, we will first determine the implications of endothelial UCP2 in diet- induced insulin resistance, a condition known to stress mitochondria. Since our preliminary data indicate that UCP2 prevents mitochondrial fragmentation, a known characteristic of obese patients with type 2 (insulin- resistant) diabetes, we will test how UCP2 impacts endothelial function in diet-induced obesity and insulin resistance. We will utilize our newly created UCP2 models with Endothelial Cell-specific KnockOut (ECKOUCP2) or Endothelial Cell-only (ECUCP2) UCP2 expression to determine the functional, morphologic, and molecular implications of endothelial UCP2 with a high-fat diet known to induce obesity and insulin resistance. We will then determine how UCP2 impacts the endothelial responses to stresses such as ischemic revascularization and tumor angiogenesis in vivo. Since our data indicate global UCP2-null mice have impaired angiogenesis with the stress of hindlimb ischemia, we will determine the specific role of UCP2 for endothelial stress in vivo. Accordingly, ECKOUCP2 and ECUCP2 mice will be tested in two models of endothelial stress: a) ischemic revascularization from hind limb ischemia and; b) tumor angiogenesis. With regards to the latter, we will also determine if acute UCP2 inhibition has the therapeutic potential to limit or shrink solid tumors. In addition to the impact on blood vessel formation, we will test endothelial UCP2 for its implications on mitochondrial mass, morphology, and network fragmentation in each model. We will also explore the role of p53 in promoting the endothelial UCP2-null phenotype since UCP2-null endothelium exhibits premature p53-dependent senescence. Finally, we will determine the functional implications of UCP2 in the endothelium. Since our data indicate UCP2 preserves endothelial function by preventing mitochondrial fragmentation, we will determine how UCP2 impacts mitochondrial function and morphology. ECs with manipulated UCP2 status will be tested mitochondrial network fragmentation and the roles of O2--mediated protein damage, mitophagy, mitochondrial biogenesis, and p53 determined in this process. We will then link the mechanism(s) of mitochondrial network fragmentation to endothelial functions relevant to angiogenesis including proliferation, migration, tube formation, and NO bioactivity. The experiments outlined above should provide us with important insight as to how UCP2 controls mitochondrial dynamics and, as a consequence, endothelial function. These insights will inform us as to how the mitochondria impact vascular homeostasis and provide us with the requisite knowledge to utilize mitochondria as a means of manipulating the endothelium in vivo and this knowledge could have wide ranging implications for wound healing, limb ischemia, and tumor metastasis.